Download The different shapes of cocci

REVIEW ARTICLE
The di¡erent shapes of cocci
André Zapun1, Thierry Vernet1 & Mariana G. Pinho2
1
Laboratoire d’Ingénierie des Macromolécules, Institut de Biologie Structurale (CEA/CNRS/UJF UMR5075), Grenoble, France; and 2Bacterial Cell Biology
Laboratory, Instituto de Tecnologia Quı́mica e Biológica, Oeiras, Portugal
Correspondence: Thierry Vernet,
Laboratoire d’Ingénierie des Macromolécules,
Institut de Biologie Structurale, (CEA/CNRS/
UJF UMR5075), 41 rue Jules Horowitz, 38027
Grenoble, France. Tel.: 133 0 4 38 78 96 81;
fax: 133 0 4 38 78 54 94;
e-mail: [email protected]
Received 26 June 2007; revised 4 October
2007; accepted 6 November 2007.
First published online February 2008.
DOI:10.1111/j.1574-6976.2007.00098.x
Editor: Jacques Coyette
Abstract
The shape of bacteria is determined by their cell wall and can be very diverse. Even
among genera with the suffix ‘cocci’, which are the focus of this review, different
shapes exist. While staphylococci or Neisseria cells, for example, are truly roundshaped, streptococci, lactococci or enterococci have an ovoid shape. Interestingly,
there seems to be a correlation between the shape of an organism and its set of
penicillin-binding proteins – the enzymes that assemble the peptidoglycan, the
main constituent of the cell wall. While only one peptidoglycan biosynthesis
machinery seems to exist in staphylococci, two of these machineries are proposed
to function in ovoid-shaped bacteria, reinforcing the intrinsic differences regarding the morphogenesis of different classes of cocci. The present review aims to
integrate older ultra-structural data with recent localization studies, in order
to clarify the relation between the mechanisms of cell wall synthesis and the
determination of cell shape in various cocci.
Keywords
bacterial division; penicillin-binding proteins;
peptidoglycan; murein.
Introduction
Most studies of cell division and morphogenesis have been
centered on the two rod-shaped laboratory workhorses:
Escherichia coli and Bacillus subtilis, due mainly to the wide
array of genetic tools available to probe the life and death of
these organisms (Errington et al., 2003; Goehring & Beckwith,
2005). Caulobacter crescentus, a bacterium that undergoes a
developmental cycle, has also emerged as a powerful model
system to investigate morphogenesis (England & Gober, 2001;
Briegel et al., 2006). However, much can be learned from
comparative studies of morphologically diverse bacteria.
Historically, determining the morphology of bacterial
cells has been an important phylogenetic tool. Yet, in the
current era of molecular phylogenetics, comparative analysis
of small subunit RNA sequences indicates that bacteria with
different morphologies exist within single branches of
phylogenetic trees and species with coccus morphology are
present in clusters with a predominantly rod morphology
(Siefert & Fox, 1998). Interestingly, once a particular lineage
exhibits coccus morphology, clusters that result from that
lineage become homogeneous for coccus morphology. This
indicates that coccal morphology, which appears to have
evolved multiple times during bacterial history, is evolutio-
FEMS Microbiol Rev 32 (2008) 345–360
narily irreversible (Siefert & Fox, 1998). The stability of
coccal shape in evolution can be due to the inherent
difficulty to regain genes for rod morphology, and/or to the
absence of selective pressure for rod shape. Genetically, it is
also easy to convert a rod into a coccus, for example by loss
(rodA, pbpA) or overexpression (bolA) of a gene (Aldea et al.,
1988; Murray et al., 1997; Henriques et al., 1998), but there
is no report of genetic alterations that convert coccal cells
into stable rod-shaped cells.
This review will address the question of the origin of
cocci, not from an evolutionary point of view, but from a
morphogenetic point of view, i.e. what determines the shape
of a coccus cell?
Before answering this question, it is useful to distinguish
between two classes of cocci: (1) organisms with truly round
cells such as pediococci, micrococci, deinococci, staphylococci or Neisseria (except Neisseria elongata), which usually
divide in either two or three alternating perpendicular
planes during consecutive division cycles, leading to arrangements in tetrads or in three-dimensional cuboidal
packets of eight cells, respectively (Fig. 1a and b); (2)
organisms whose cells are elongated ellipsoids, such as
enterococci, streptococci or lactococci (and some other
genera such as Lactovum, Leuconostoc, Weissella and
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346
A. Zapun et al.
(a)
debated (Tiyanont et al., 2006) and although the key
effectors of cell wall biosynthesis controlled by MreBs have
not yet been identified.
A second cytoskeleton element is composed of the
tubulin homologue FtsZ, which polymerizes as a ring at the
division site and is widely distributed in bacteria (Errington
et al., 2003; Goehring & Beckwith, 2005). The recruitment of
other division proteins in Escherichia coli and B. subtilis is
subordinated to the presence of FtsZ at the division site.
FtsZ has therefore been considered to be the prime organizer
of the division process, onto which other components are
assembled to form the so-called divisome (Buddelmeijer &
Beckwith, 2002; Errington et al., 2003). Among the later
division proteins that are recruited at the division site is FtsI,
one of the penicillin-binding proteins (PBPs). These enzymes are responsible for the assembly of peptidoglycan, the
main component of the bacterial cell wall (Sauvage et al.,
2008). FtsZ-directed cell wall synthesis at the division site of
rod-shaped bacteria results in the formation of the division
septum, which, after cell division is completed, is converted
into the new pole of each of the two daughter cells.
The presence in rods of the two families of cytoskeleton
proteins, MreB and FtsZ, is therefore associated with the
two main phases of cell wall growth: the elongation and
the division, respectively. MreB proteins are not encoded in
most genomes of cocci. The few exceptions include some
cyanobacteria and the Chlamydia, which have a spherical
shape but have the ability to undergo morphological differentiation during their life cycle (Carballido-Lopez, 2006).
Thus, the truism of this section’s title, the absence of
cylindrical side-wall in cocci, may find its molecular basis
in the absence of an actin-like cytoskeleton, which precludes
a true elongation. In cocci, FtsZ-dependent cell wall synthesis is therefore predominant and determinant for morphogenesis, where it can account for the synthesis of the entire
new hemisphere of each daughter cell.
FtsZ-dependent cell wall synthesis at the division site
can be observed in Staphylococcus aureus, by visualization of
peptidoglycan synthesis using fluorescent vancomycin, under conditions where it binds only to pentapeptides that are
present on the new peptidoglycan or its precursor (Pinho &
Errington, 2003). This method has shown that staphylococcal cell wall synthesis occurs mainly, if not exclusively, at the
division site. However, if FtsZ is depleted from Staphylococcus aureus cells, the septum no longer forms and cell wall
synthesis becomes delocalized over the entire surface of the
cell, allowing it to enlarge up to eight times its normal
volume, before lysing (Pinho & Errington, 2003).
Ovococci also synthesize the cell wall mostly at the
division site, with the new hemispheres of the daughter cells
being synthesized between the two parting old hemispheres
and, accordingly, labeling of Streptococcus pneumoniae with
fluorescent vancomycin shows that peptidoglycan synthesis
(b)
(c)
Fig. 1. Bacterial division over successive division cycles in (a) two
perpendicular planes (e.g. Neisseria or pediococci), (b) three perpendicular planes (e.g. staphylococci) or (c) one parallel plane (e.g. enterococci
or streptococci).
Oenococcus, Melissococcus, Vagococcus), which divide in
successive parallel planes, perpendicular to their long axis
(Fig. 1c). These can be observed as isolated cells, diplococci
or small chains, depending on the degree of cell separation.
To the authors’ knowledge, there is no widely accepted
term to distinguish ellipsoid bacteria from spherical cocci and
therefore the term ovococcus is proposed to designate these
bacteria with ovoid shape. Note that this designation is only
morphological, as common shape is not strictly correlated
with evolutionary relationship. In this review, mainly staphylococci will be considered as a model for spherical cocci and
enterococci and streptococci as models for ovococci.
Lack of cylindrical elongation in cocci
Bacterial morphology was traditionally assumed to be
determined by the cell wall, which acts as an external
scaffolding or exoskeleton. This was based on the facts that
isolated cell wall sacculi retained the specific shape of a
particular cell (Höltje, 1998) and that most mutations
originally described as affecting cell shape were located in
genes involved in cell wall synthesis (Spratt, 1975; Tamaki
et al., 1980; Honeyman & Stewart, 1989; Henriques et al.,
1998). Recently, an internal helical scaffolding or cytoskeleton was identified in rod-shaped bacteria, which is
composed of actin homologues – encoded by mreB and
mreB-like genes – that have a direct role in cell shape
determination. These internal scaffolds, rather than behaving like rigid skeletons, may determine bacterial shape by
directing peptidoglycan-synthesizing machineries involved
in elongation of the cell, which in turn construct the cell’s
peptidoglycan exoskeleton (Daniel & Errington, 2003; Dye
et al., 2005; Carballido-Lopez, 2006), although this is still
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FEMS Microbiol Rev 32 (2008) 345–360
347
Coccal morphogenesis
occurs mostly at mid cell (Daniel & Errington, 2003; Ng
et al., 2004), confirming the previous pulse chase experiments that did not directly monitor peptidoglycan incorporation, but rather the incorporation of additional
components of the cell wall, such as protein antigens or
teichoic acids, which are attached to the peptidoglycan
(Cole & Hahn, 1962; Tomasz et al., 1975). FtsZ-depletion
studies have not been carried out in ovococci. However,
colocalization of all high-molecular-weight (HMW) PBPs
with FtsZ at the onset of division is consistent with an FtsZtriggered cell wall synthesis. Also, zantrins, which are small
molecules that perturb FtsZ function, inhibit division in
Streptococcus pneumoniae, resulting in cell enlargement
(Margalit et al., 2004).
One or two types of division-specific cell
wall synthesis in cocci
Previous ultrastructural studies using Staphylococcus aureus,
carried out primarily by Giesbrecht et al. (1998), have shown
that the synthesis of the septum proceeds by centripetal
growth resembling a closing iris, until the inner edge of the
growing septum eventually fuses in the center of the cell.
After the septum or cross-wall is complete, the two daughter
cells are still held together within a single sphere, whose cell
wall is a homogeneous-looking structure, with no signs of
invagination (Fig. 2a). Only at the end of cell division is the
septum split into two surfaces that become the new hemispheres of each of the daughter cells (Giesbrecht et al., 1998).
Recently, cryo-electron microscopy of frozen-hydrated thin
sections of Staphylococcus aureus revealed a differentiated
cell wall at the septum with two zones of high density, which
correspond to two adjacent cross-walls, located between two
low-density zones, and separated by a third zone of lowdensity (Matias & Beveridge, 2007). The two low density
zones adjacent to the cell membrane appear as an extension
of the periplasmic space described recently in a number of
gram-positive bacteria, including the cocci Enterococcus
gallinarum, Streptococcus gordonii and Staphylococcus aureus
(Matias & Beveridge, 2006; Zuber et al., 2006). The lowdensity region between the two cross-walls could correspond to a highly fragile septal region that would facilitate
or result from the action of the autolysins responsible
for splitting of the septum (Matias & Beveridge, 2007). After
splitting, some remodeling of the peptidoglycan may occur
when the flat septum becomes spherical, but this alteration
of conformation could result merely from exposure of the
new hemisphere to high internal osmotic pressure, immediately after cell separation.
When a second and third round of division occur in
Staphylococcus aureus, the septum is synthesized in consecutive perpendicular planes, similar to the mode of division of
Sarcina or some species of cyanobacteria Synechocystis
FEMS Microbiol Rev 32 (2008) 345–360
Fig. 2. Electron micrographs of cocci. (a) Thin section of Staphylococcus
aureus spherical cells that have completed septum formation. (b) Longitudinal thin section of dividing ovococcus Streptococcus pneumoniae R6.
(Giesbrecht et al., 1998) (Fig. 1b). This was initially inferred
from scanning electron microscopy results, which showed
regular cuboidal packets of eight Staphylococcus aureus cells
most likely resulting from three divisions along orthogonal
planes (Tzagoloff & Novick, 1977). However, under the light
microscope, Staphylococcus aureus appears as clusters of cells
without an obvious geometric arrangement. This is probably due to the activity of lytic enzymes responsible for the
splitting of the division septum that seem to cause a
postfissional movement of the cells, leading to the formation
of irregular clusters (Koyama et al., 1977). Despite the fact
that these observations were made three decades ago, the
mechanism that determines the precise placement of the
septum in alternating perpendicular planes is far from
understood.
A detailed description of the growth and division process
of ovococci was provided in an impressive series of ultrastructural papers from the 1970s by Gerry Shockman,
Michael Higgins and colleagues on the morphogenesis of
Enterococcus hirae ATCC 9790, as well as early work on
Streptococcus pneumoniae by Alexander Tomasz. These landmark studies, based mostly on electron microscopy of
negatively stained thin sections, showed that ovococcal cells
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348
are surrounded in their middle by an annular outgrowth of
peptidoglycan, often referred to as the equatorial ring
(Tomasz et al., 1964; Higgins & Shockman, 1970). The first
cell wall-related event in cell division is the appearance of a
small ingrowth below the equatorial ring. The equatorial
ring is then split, more or less in its middle, and the two
resulting rings separate while a new peripheral cell
wall appears in between. The small annular ingrowth
remains equidistant from the two new parting equatorial
rings, and at some point starts to grow centripetally to form
a septal disc. The septal disc undergoes gradual and simultaneous closure at its center and splitting at its periphery
(Fig. 2b).
The first model for the growth of ovococci envisioned a
single site of centripetal septal synthesis, with gradual
splitting of the septum feeding the peripheral growth
(Higgins & Shockman, 1970). However, careful measurements of the septal and peripheral wall surface area showed
that the latter was growing faster than allowed by septal
splitting alone. A second model was proposed, which
included a second site of peptidoglycan synthesis (Higgins
& Shockman, 1976). This peripheral synthesis was supposed
to occur diffusely along a gradient, with the greatest activity
near the site of septal splitting.
The idea of ovococci having two types of division-specific
cell wall synthesis, and spherical cocci only one, was later
supported by extensive mutagenesis and drug treatment studies
by Satta et al. (Higgins et al., 1974; Gibson et al., 1983; Lleo
et al., 1990). They found that temperature-sensitive mutants,
which grow longer and fail to septate at a nonpermissive
temperature, could be isolated from most ovococcal species
investigated. These species underwent the same morphological
changes upon treatment with antibiotics supposed to block
only septation. In contrast, no longitudinal growth was
obtained from truly spherical cocci such as staphylococci or
certain Neisseria species, either by mutation or with drugs
(Higgins et al., 1974; Gibson et al., 1983; Lleo et al., 1990). In
Enterococcus hirae, treatment with penicillin at division-inhibiting concentrations led to the formation of long filaments under
some growth conditions of temperature and medium (Fontana
et al., 1983). The concept of two competing sites of peptidoglycan synthesis in ovococci was then introduced, based on the
observation that suppression of septation apparently led to
unchecked longitudinal expansion (Higgins et al., 1974; Gibson
et al., 1983; Lleo et al., 1990).
Interestingly, Streptococcus mutans displays an odd behavior regarding the morphology of some strains as its length
to width ratio can span values from 1 to 5 due to variations
of the ratio of K1/bicarbonate in the growth medium (Tao
et al., 1988). As pointed out by the authors, the morphological variations exhibited by some strains of Streptococcus
mutans would be consistent with the two-site model of cell
wall synthesis, where the relative activity of the peripheral
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A. Zapun et al.
and septal synthesis, and/or the timing of septation, would
be influenced by the ratio of some ions (Tao et al., 1993).
Following the terms introduced by Higgins & Shockman
(1976), these types of synthesis shall be called as septal for
the formation of the cross-wall, perpendicular to the main
axis of the cell, and peripheral for the longitudinal component. Spherical cocci have only septal synthesis. Note that
both septal and peripheral cell wall synthesis are considered
as division specific, and that peripheral synthesis is distinct
from elongation of rod-shaped bacteria.
However, besides septal and elongation modes of cell
wall synthesis, rods also seem to have an additional mode
of lateral cell wall synthesis, less well characterized, which
occurs in the periphery of the cell, near mid cell, but is
dependent on FtsZ. Evidence comes from the observation of
areas of high, FtsZ-dependent, peptidoglycan synthesis at
the division site of Escherichia coli, even when septal synthesis is specifically suppressed, for example by inactivation of
the cell division-specific transpeptidase (Wientjes & Nanninga, 1989; de Pedro et al., 1997). The existence of a
significant time gap between FtsZ ring formation and
assembly of the septal peptidoglycan synthesis machinery
in Escherichia coli raised the possibility that preseptal
peptidoglycan synthesis could occur during that period
(Aarsman et al., 2005). Furthermore, PBPs known to
participate in the elongation of Escherichia coli and B.
subtilis were found at the division site of these rod-shaped
bacteria (Den Blaauwen et al., 2003; Scheffers et al., 2004).
In Escherichia coli, the preseptal synthesis was found to be
‘penicillin’-insensitive. Indeed, a number of tested b-lactams
did not inhibit this FtsZ-dependent synthesis, including
drugs specific of class A PBPs or of the elongation-specific
PBP2. These observations suggested that a monofunctional
glycosyltransferase of Escherichia coli may participate in the
preseptal synthesis (Aarsman et al., 2005). Alternatively, not
all the HMW PBPs of Escherichia coli may have been
inhibited with the tested drugs.
In C. crescentus, this mode of cell wall synthesis at the mid
cell is more evident and was shown to be dependent on FtsZ
but independent of MreB and to occur before constriction
of the cell takes place (Aaron et al., 2007). Thus, it is
speculated that the division of rods includes the same two
types of synthesis as ovococci: peripheral and septal, both
FtsZ dependent. In addition to these division-specific
modes of cell wall assembly, rods have MreB, dependent
elongation, which is absent from cocci.
Similar sets of PBPs for similar shapes -one or two machineries for peptidoglycan
assembly
PBPs are the enzymes responsible for the synthesis of long
chains of tandemly repeated disaccharide units that make up
FEMS Microbiol Rev 32 (2008) 345–360
349
Coccal morphogenesis
(a)
PBP1
PBP2
(b)
PBP2b
PBP2x
DNA
merged
bright
field
Fig. 3. Localization of PBPs in cocci. (a) Septal localization of PBP1 (localized by immunofluorescence) and PBP2 (localized using an N-terminal green
fluorescent protein fusion), the two essential PBPs of Staphylococcus aureus. (b) Immunofluorescence microscopy of dividing Streptococcus
pneumoniae cells. PBP2b was revealed with mouse antiserum and Cy3-coupled goat anti-mouse antibodies. PBP2x was revealed with rabbit antiserum
and Cy2-coupled goat anti-rabbit antibodies. DNA was diamidino-2-phenylindole stained. Images are raw. The scale bar is 1 mm.
the glycan strands of the peptidoglycan, as well as for their
cross-linking via peptide bridges. They can be classified as
HMW class A PBPs, which include bifunctional proteins
that have both glycosyl transferase activity (for the synthesis
of the glycan strands) and transpeptidase activity (for the
cross-linking of the peptidoglycan); HMW class B PBPs,
which include proteins that have a N-terminal domain with
unknown function and a C-terminal transpeptidase
domain; and low molecular weight (LMW) PBPs, which
usually have carboxypeptidase or endopeptidase activity
(Goffin & Ghuysen, 1998). b-Lactam antibiotics such as
penicillin inhibit the transpeptidase, carboxypeptidase or
endopeptidase activities.
The number of PBPs varies considerably among bacteria,
with the rod-shaped model organisms Escherichia coli and B.
subtilis having 12 and 16 PBPs, respectively, while cocci tend
to have a lower number, usually from four to seven PBPs.
These numbers were initially determined by incubating cells
or membranes with radio-labeled penicillin before gel
electrophoresis. This procedure often revealed band patterns
that are difficult to correlate with known pbp genes present
in the sequenced genomes. Although the presence of addiFEMS Microbiol Rev 32 (2008) 345–360
tional PBPs in some strains cannot be ruled out, the
detection of unexpected bands can most easily be explained
by various proteolytic cleavages of known PBPs (Coyette
et al., 1980). The fact that many PBPs have redundant
functions in an organism has made it difficult to assign a
specific function for each PBP.
It has been suggested that PBPs work in multi-enzymatic
complexes that would include not only peptidoglycan
synthetic enzymes but also peptidoglycan-degrading or lytic
enzymes, in order to co-ordinate both processes, preserving
the integrity of the cell, and that different machineries would
be responsible for each mode of peptidoglycan synthesis
(Höltje, 1996, 1998).
Staphylococcus aureus cells have only four PBPs. The two
essential staphylococcal PBPs (HMW class B PBP1 and
HMW class A PBP2, the only bifunctional PBP in Staphylococcus aureus) localize at the division site (Fig. 3a) (Pinho &
Errington, 2005; Pereira et al., 2007). PBP4, a LMW PBP,
also seems to localize at that place (P.M. Pereira and M.G.
Pinho, unpublished data). The localization of PBP3 (HMW
class B) has not yet been defined, but it seems unlikely that it
would catalyze de novo cell wall synthesis around the entire
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350
A. Zapun et al.
(a)
(b)
I
II
Fig. 4. Proposed models for cell wall assembly in spherical cocci (a) and ovococci (b). The parental cell wall is in black; the new cell wall of peripheral and
septal origin is in dark or light gray, respectively. Peripheral and septal peptidoglycan synthesis machineries are in red and green, respectively. Black
contours indicate active machineries. The splitting machinery is in cyan, with a jaw to indicate activity. In pathway I (b), both machineries are localized at
the leading edge of the septum, but are functioning successively, while in pathway II, the peripheral machinery remains at the periphery of the septal
disc and contributes with material to the inner face of the cell wall.
periphery of the cell without the assistance of a HMW class
A PBP. Furthermore, a PBP3 mutant has no major observable morphological defects (Pinho et al., 2000). The small
number of PBPs and their septal localization is in accordance with the fact that Staphylococcus aureus synthesizes
the cell wall at the division site using one septal synthetic
machinery (Fig. 4a), although it cannot be excluded that
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another type of synthesis involving the second, nonessential,
HMW class B PBP3 may exist. Methicillin-resistant Staphylococcus aureus (MRSA) strains have acquired an additional
PBP – PBP2A – which has a transpeptidase domain with a
very low affinity for b-lactam antibiotics and, together with
the transglycosylase domain of PBP2, catalyzes cell wall
synthesis in the presence of a high concentration of
FEMS Microbiol Rev 32 (2008) 345–360
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Coccal morphogenesis
antibiotics (Pinho et al., 2001). The localization of this
protein as well as its activity in the absence of antibiotics
remains unknown.
PBPs from other spherical cocci have not been localized,
but the fact that Neisseria meningitidis or Neisseria gonorrhoeae have only four described PBPs (Nolan & Hildebrandt, 1979; Barbour, 1981; Stefanova et al., 2004), which
include only one representative of HMW class A (PBP1) and
one of HMW class B (PBP2) PBPs, may indicate that these
species also have only one cell wall synthetic machinery.
As mentioned earlier and in contrast to spherical cocci,
ovococci have two types of division-specific cell wall synthesis, which are necessary for their specific ovoid shape. To
achieve this ovoid shape, a common set of PBPs may be
required (Table 1), which includes three class A (PBP1a, 1b
and 2a) and two class B (PBP2b and 2x) PBPs. A LMW PBP
(PBP3) has carboxypeptidase activity that trims the free end
of peptidoglycan pentapeptides, which then become of
limited use for the transpeptidation reaction, as they can
only serve as acceptors.
Enterococci possess a supplementary class B PBP (PBP5),
with a low affinity for penicillins, which confers an intrinsic
resistance to these antibiotics. However, in the absence of
penicillins, this PBP can be deleted without notable consequences on the peptidoglycan composition or cell morphology (Sifaoui et al., 2001). A true exception to the
uniform number of basic PBPs in ovococci is the absence of
PBP2b in Streptococcus pyogenes, which will be discussed
below. Others are the presence in Streptococcus pyogenes or
Streptococcus agalactiae of one or two additional genes
coding for uncharacterized LMW PBPs.
The common set of PBPs present in ovococci may be
organized in two cell wall synthetic machineries, which
would catalyze the two modes of division-specific cell wall
synthesis. Based on data from immunofluorescence microscopy of Streptococcus pneumoniae, an initial model was
proposed, in which two cell wall synthesis machineries
would have different localizations (Morlot et al., 2003), but
later proved to be incorrect, as shown below. New data
obtained with better batches of antisera against some PBPs
have revealed that all the PBPs have the same localization
at mid cell, at the resolution of the immunofluorescence
microscopy, throughout the cell cycle (Fig. 3b). Is it possible
to reconcile these new data showing that all the PBPs are
colocalized, with the existence of two modes of synthesis,
implied by the ultrastructural, mutational and drug treatment studies (Higgins & Shockman, 1976; Lleo et al., 1990)?
Here, a revised model is proposed that accommodates two
machineries of peptidoglycan synthesis with a single localization (Fig. 4b). The machinery responsible for the peripheral component of cell wall synthesis would start to add
material at mid cell, on the inner face of the cell wall, with
the concomittant splitting of the new wall, resulting in the
FEMS Microbiol Rev 32 (2008) 345–360
observed initial phase of longitudinal growth. A second
phase would start somewhat later, with the activity of a
second machinery, to build the septal cross-wall. Septal
assembly of the peptidoglycan should occur faster than
septal splitting to produce the observed cross-wall. The two
types of synthesis could occur strictly successively, or
partially simultaneously during the septal phase. After
closure of the septum, splitting goes on to separate the
daughter cells. Both machineries could be located at the
leading edge of the closing septum, but activated at different
points of the division process. Alternatively, the machinery
for peripheral synthesis might not follow the leading edge of
the septum, but remain at its periphery. Thus, once septum
formation starts, the peripheral machinery might continue
adding material on the inner face of the cell wall. This
model, which is essentially that proposed thirty years ago by
Higgins and Shockman, can account for the measured
thickening of the cell wall near the splitting site (Higgins &
Shockman, 1976; Lleo et al., 1990).
Other proteins involved in cell wall
synthesis of cocci
In addition to PBPs, the synthesis of peptidoglycan is likely
to require the presence of shape, elongation, division and
sporulation (SEDS) proteins (Henriques et al., 1998). SEDS
proteins are integral membrane proteins, with 10 membrane-spanning segments (Gerard et al., 2002), which presumably participate in peptidoglycan synthesis, as implied
by the frequent organization in operons of the genes for
SEDS proteins and class B PBPs, and the identical phenotypes resulting from deletion of class B PBPs or their cognate
SEDS protein (de Pedro et al., 2001; Thibessard et al., 2002).
Although the biochemical function of these proteins remains unknown, it has been proposed (but never experimentally shown) that SEDS proteins might be the flippases
required to translocate the peptidoglycan precursors from
the cytoplasm, through the plasma membrane, to the
extracellular site (Ehlert & Holtje, 1996; Ghuysen & Goffin,
1999). The phenotypes of FtsW mutants and RodA depletion in rod-shaped bacteria indicate that FtsW is the SEDS
protein involved in division, whereas RodA is required for
elongation (e.g. Ishino et al., 1989; Boyle et al., 1997;
Henriques et al., 1998).
Interestingly, Staphylococcus aureus has two SEDS proteins, despite apparently having only septal cell wall synthesis. The prediction is that FtsW would work with the
essential class B PBP1 within the main septal machinery of
cell wall synthesis. Correspondingly, staphylococcal RodA
would play a minor role, working with the nonessential class
B PBP3. Neisseria lack RodA, in accordance with the
presence of a single HMW class B PBP.
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SAG0159
Spy0097
SpyM3_0074
M6_Spy0130
gbs0288
SAG0298
Spy1649
SpyM3_1390
M6_Spy1401
M6_Spy1753
SpyM3_1758
Spy2059
SAG2066
gbs2020
SMU.1949
M28_Spy1739
SMU.1991
SMU.467
stu0212
M28_Spy0080
stu1869
stu0230
str0212
M28_Spy1396
str1869
str0230
spr1823
SpyM18_2120
spr1909
spr0329
SP2010
SpyM18_0098
SP2099
ponA SP0369
pbpF EfaeDRAFT_
2538
LL2108
SpyM18_1661
pbpZ EfaeDRAFT_
0375
LL0386
ponA
EfaeDRAFT_0179
ponA LL0543
EF0680
PBP2a
SAG0765
gbs0785
SMU.597
stu0613
str0613
penA spr1517
penA SP1673
pbpA EfaeDRAFT_
1462
LL0339
EF2857
M28_Spy1408
SpyM18_1674
M6_Spy1413
SpyM3_1401
pbpX ftsI Spy1664
pbpX SAG0287
gbs0277
SMU.455
stu1701
str1701
pbpX spr0304
pbpX SP0336
pbpB EfaeDRAFT_
2224
pbpX LL0867
PBPC EF0991
PBP2x
PBP2b
EF1740
EF1148
PBP1b
PBP1a
PBP5
pbp5 EfaeDRAFT_
0894
PBP4 EF2476
dacA SpyM18_
0280
dacA2 M28_Spy0243
dacA SpyM3_
0214
M6_Spy0279
dacA Spy0292
SAG0147
gbs0143
SMU.253
dacA1 stu0080
dacA1 str0080
spr0776
SP0872
dacA LL2263
Contig588
dacA EF3129
PBP3
DD-Carboxypeptidase
SpyM18_
0279
dacA1 M28_Spy0242
M6_Spy0278
SpyM3_0213
SAG0146
gbs0142
Locus names of the various sequencing projects are given as well as alternative names found in the literature or databases. Genomes were searched by nucleotide and protein BLAST.
Enterococcus faecalis
V583
Enterococcus
faecium DO
Lactococcus
lactis IL1403
Streptococcus
pneumoniae
TIGR4
Streptococcus
pneumoniae R6
Streptococcus
thermophilus
CNRZ1066
Streptococcus
thermophilus
LMG18311
Streptococcus
mutans UA159
Streptococcus
agalactiae
NEM316
Streptococcus
agalactiae
2603V/R
Streptococcus
pyogenes
SF370
Streptococcus pyogenes
MGAS315
Streptococcus
pyogenes MGAS10394
Streptococcus
pyogenes MGAS8232
Streptococcus
pyogenes MGAS6180
ClassB (monofunctional)
Class A (bifunctional)
Table 1. Presence of PBPs in ovococci with fully sequenced genome
SpyM18_
1051
M28_
Spy0794
M6_Spy0815
SpyM3_1390
Spy1093
352
A. Zapun et al.
FEMS Microbiol Rev 32 (2008) 345–360
353
Coccal morphogenesis
Class A
PBP
?
PBP2b
Class A
PBP
?
DivIB
PBP2x
FtsL
DivIC
MreC
FtsW
RodA
MreD
Synthesis
Regulation ?
Peripheral machinery
Synthesis
Regulation ?
Septal machinery
Fig. 5. Established and putative proteins participating in the peripheral and septal peptidoglycan synthesis machineries of ovococci. The identity of the
bifunctional class A PBPs participating in each phase remains unknown.
Ovococci also possess two SEDS proteins. In Streptococcus
pneumoniae, FtsW was found to have the same localization
at mid cell as the PBPs (Morlot et al., 2004a). The pbp2x
gene is downstream of ftsL as is ftsI in most genomes
(Massidda et al., 1998), indicating that PBP2x is likely the
class B PBP participating in the septal machinery with FtsW
(Fig. 5). To further support this identification, PBP2x and
PBP2b from Streptococcus pneumoniae show better similarity to PBP3 (FtsI) and PBP2 in Escherichia coli, respectively.
PBP2b and RodA would therefore be part of the peripheral
machinery (Fig. 5).
Streptococcus pyogenes, which lacks both PBP2b and
RodA, often appears less elongated and more spherical than
other ovococci. It is conceivable that it performs only septal
synthesis, giving rise to cells lacking the longitudinal component provided by the peripheral synthesis. In support of
this theory is the observation that opaque colony variants of
Streptococcus pyogenes, which apparently fail to split their
cross-wall, form stacks of flattened cells with very little nonseptal wall (Swanson & McCarty, 1969). Mutants of Streptococcus thermophilus defective in RodA or PBP2b are also
more spherical than the parental strain, with a longitudinal
cellular length about half that of the wild type (Stingele &
Mollet, 1996; Thibessard et al., 2002). In Streptococcus
gordonii, the deletion of PBP2b results in more complex
morphological defects with aberrant septa in some cells and
an increased susceptibility to lysis (Haenni et al., 2006).
Besides PBPs and SEDS proteins, other proteins may
contribute to cell wall construction. Among division proteins, DivIB(FtsQ), FtsL and DivIC(FtsB) are bitopic membrane proteins, with their major domains being extracellular
(Fig. 5) (Errington et al., 2003; Goehring & Beckwith, 2005).
The gene encoding DivIB is often in an operon with genes
involved in the synthesis of peptidoglycan precursors. The
FEMS Microbiol Rev 32 (2008) 345–360
gene coding for FtsL is often adjacent to that encoding the
septal class B PBP. The three genes are completely absent
from wall-less bacteria (Margolin, 2000). These arguments
suggest that the unknown functions of DivIB, FtsL and
DivIC are related to cell wall formation. FtsL and DivIC are
small proteins. Sequence-based predictions indicate that the
extracellular domains adopt essentially a coiled-coil conformation. The extracellular region of DivIB appears to consist
of two autonomously folding domains (Robson & King,
2006). A complex of the extracellular domains of the three
proteins from Streptococcus pneumoniae was reconstituted
in vitro, provided that heterodimerization of the extracellular domains of FtsL and DivIC was artificially constrained,
presumably to compensate for the deletion of the transmembrane segment (Noirclerc-Savoye et al., 2005). A similar complex is formed in Escherichia coli (Buddelmeijer &
Beckwith, 2004). In vivo, the three proteins appeared to
colocalize at the division site during septation.
What could be the function of DivIB, FtsL and DivIC?
Assuming that they function in cell wall assembly, this role
may be restricted to the division, as no paralogues exist for
the elongation synthesis of rod-shaped bacteria. Therefore,
the function of these three proteins may be found in features
that distinguish division- and elongation-specific peptidoglycan synthesis. Two such characteristics come to mind.
Firstly, the elongation of bacilli produces a cylinder; the
amount of cell wall generated at different stages of the
formation of a cylinder is constant. In contrast, the amount
of synthesis required during division diminishes progressively until extinction, as the septal hole closes. DivIB, FtsL
and DivIC might intervene in the regulation of this diminishing synthesis. A second difference between the septal and
the lateral cell wall is that the lateral wall is subjected to
osmotic pressure, whereas the septal cross-wall is not. In the
2008 Federation of European Microbiological Societies
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c
354
absence of osmotic pressure, septal synthesis might require
an additional scaffolding function, which could be provided
by DivIB, FtsL and DivIC.
MreC and MreD may also be key proteins involved in cell
wall synthesis that have not been well studied in cocci yet.
MreC, a bitopic membrane protein with a major extracellular domain, and MreD, an integral membrane protein
(Fig. 5), are both involved in the elongation phase of rodshaped bacterial morphogenesis, by interacting with Mbl
and MreB in B. subtilis, and MreB in Escherichia coli, which
lacks Mbl (reviewed by Stewart, 2005). Mbl is a cytoskeleton
actin-like protein from the MreB family. Through the MreB/
Mbl connection, MreC and MreD are somehow involved in
the control of peptidoglycan synthesis (Leaver & Errington,
2005). As both MreB and Mbl are missing in cocci, it will be
of interest to find out whether MreC and MreD have a role
in peptidoglycan synthesis by themselves. In ovococci, it is
suggested that MreC and MreD might take part in peripheral synthesis (Fig. 5). Deletion of MreD in Streptococcus
thermophilus produced smaller cells (Thibessard et al.,
2004). Note that MreC and MreD are absent in Streptococcus
pyogenes and Streptococcus agalactiae. Interestingly, these
two species have additional LMW PBPs. This correlation
may be coincidental or may indicate that two different
solutions have evolved to regulate some aspects of murein
metabolism in ovococci.
How do cell wall synthesis machineries
localize? Co-ordinating cell wall synthesis
and the cell cycle
To produce cells of proper shape and size, the activity and
localization of the cell wall synthesis and degradation
machineries must be correctly regulated. Most importantly,
septal cell wall formation during division must be coordinated with the other processes of cell division, such as
membrane invagination, DNA replication or chromosome
segregation, to avoid catastrophic breakage of the chromosome by division through the nucleoid, and must occur
exactly at the middle of the cell to ensure the generation of
two identical daughter cells.
Early investigations of Enterococcus hirae by Higgins’s
group showed that initiation of division, defined as the
duplication of the equatorial ring, occurs at constant cell
volume, independently of the growth rate (Gibson et al.,
1983). Thus, at a fast growth rate, a second round of division
can start, before closure of the former septum. Using
inhibitors of DNA synthesis and cell shape analysis, initiation of cell wall formation (peripheral and septal) was
shown to be independent of the end of chromosome
replication, but closure of the septum was not (Higgins
et al., 1974; Gibson et al., 1983; Lleo et al., 1990). How cells
2008 Federation of European Microbiological Societies
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c
A. Zapun et al.
sense their volume and signal the start of division is entirely
unknown.
However it is known that the initiation of division
requires the formation of a properly localized FtsZ ring.
Rod-shaped bacteria have two main systems to ensure that
the FtsZ ring assembles at mid cell: the nucleoid occlusion
effect and the Min system.
The nucleoid occlusion effect, first postulated by Woldringh
et al. (1990), relies on the ability of the nucleoid, the bacterial
equivalent of the nucleus, to prevent division in its vicinity.
Recently Noc, a specific effector of nucleoid occlusion, was
identified in B. subtilis (Wu & Errington, 2004), and a
functional analogue, SlmA, was identified in Escherichia coli
(Bernhardt & de Boer, 2005). The Noc protein is a nonspecific
DNA-binding protein that prevents the division machinery
from assembling in the vicinity of the nucleoid. The Min system
acts to prevent the formation of septa near the cell poles (for a
recent review see Lutkenhaus, 2007). MinC and MinD proteins
form a complex that acts as an antagonist of the FtsZ ring
assembly. In B. subtilis, the activity of MinCD is restricted to the
vicinity of the poles by the topological specificity factor DivIVA,
which directly recruits the inhibitor to that place. In Escherichia
coli, MinCD oscillate between the poles, giving rise to a gradient
with the lowest concentration at the middle of the cell, and
MinE is the topological marker that restricts the activity of
MinCD at the middle of the cell. The net effect of both systems
is that polymerization of FtsZ in rod-shaped bacteria occurs
exclusively at the only place that results in the division of a
cylinder with a constant diameter in two equal daughter cells,
i.e., the middle of the cell, and it occurs when this place has
already been cleared from the nucleoid.
Min proteins are present in various species of cocci such
as N. gonorrhoeae, N. meningitidis, Synechocystis sp. or
Deinococcus radiodurans but are missing from other cocci,
such as Staphylococcus, Enterococcus or Streptococcus. So how
do these cocci find their middle? Contrary to rod-shaped
bacteria, spherical cocci have not one, but an infinite
number of potential cell division planes that can give rise to
two equal daughter cells, as the middle of the cell can be
defined by any circumference with maximum diameter.
Therefore, although the existence of a protein-based system
for the selection of the division site in cocci cannot be ruled
out, spherical cells may not need a Min-like mechanism to
determine the future division site. The zone of maximum
diameter is likely to be the site where a circular polymer
lying against the cell membrane, such as the FtsZ ring,
would be most stable. However, from the infinite number of
division planes that divide a coccus in to two identical cells,
only a few will not overlap the segregating chromosomes.
Round cocci divide into two (e.g. Neisseria) or three (e.g.
Staphylococcus) perpendicular division planes. Therefore,
the chromosome also has to segregate along two or three
perpendicular axes that are perpendicular to the septal
FEMS Microbiol Rev 32 (2008) 345–360
355
Coccal morphogenesis
planes. The mechanism that determines the choice of axis
for chromosome segregation and plane of division remains
totally unknown.
Ovococci always divide in the same plane, perpendicular
to the longer axis of the cell. However, they also miss the Min
system and nucleoid occlusion does not seem to operate in
these bacteria. Indeed, the equator, where most division
proteins are found at the start of division in Streptococcus
pneumoniae, is located precisely around the nucleoid (Morlot et al., 2003, 2004a, b). In these organisms, equatorial ring
duplication ensures that the division site is always marked.
This equatorial ring marks the cellular site with the largest
circular perimeter, which, as mentioned above, may be the
most stable place for the FtsZ ring, therefore possibly
circumventing the requirement for a Min-like system for
determination of the division site. Interestingly, in Streptococcus pneumoniae, the division proteins are always localized,
mostly at mid cell and sometimes at the poles (Morlot et al.,
2003, 2004a, b). This is in marked contrast with the behavior
of division proteins in rods, as in Escherichia coli and B.
subtilis the division site is not marked during the elongation
phase but instead requires the complex nucleoid occlusion
and Min systems to be defined at the time of division.
It is worth mentioning that although staphylococci and
streptococci lack MinCD, they have DivIVA. In B. subtilis,
DivIVA appears to be involved not only in the positioning of
the division site through a functional interaction with the
MinCD system but also in chromosome segregation
during sporulation (Marston & Errington, 1999; Wu &
Errington, 2003).
In Streptococcus pneumoniae, a DivIVA deletion mutant
shows division and morphological defects, with aberrant
shapes and incomplete septa, as well as some cells devoid of
nucleoid, indicating problems in chromosome segregation
(Fadda et al., 2003). A similar phenotype was observed in
Enterococcus faecalis following disruption of divIVA (Ramirez-Arcos et al., 2005). In contrast, in Staphylococcus aureus,
deletion of DivIVA resulted in no distinctive phenotype
(Pinho & Errington, 2004). In Streptococcus pneumoniae,
DivIVA is found at the division site and the poles, whereas in
Staphylococcus aureus, DivIVA is mainly at the septum
(Pinho & Errington, 2004; Fadda et al., 2007). Results from
two-hybrid experiments in Escherichia coli indicate that
pneumococcal DivIVA interacts with many proteins of the
division machinery (Pinho & Errington, 2004; Fadda et al.,
2007), but it is still unclear whether the morphological
defects resulting from the deletion of DivIVA are due to a
direct role of DivIVA in the control of cell wall synthesis, or
due to a more general function in cell division.
Another feature that may mark the division site is a local
difference in the lipid composition of the cell membrane. In
Escherichia coli and B. subtilis, cardiolipin was found to be
concentrated at the division site (Mileykovskaya & Dowhan,
FEMS Microbiol Rev 32 (2008) 345–360
2000; Koppelman et al., 2001; Kawai et al., 2004). The same
is true of phosphatidylethanolamine and several lipid synthesis enzymes in B. subtilis (Nishibori et al., 2005). Interestingly, the drug cerulenin, which inhibits acyl carrier protein
synthases (enzymes that mature proteins involved in fatty
acid synthesis), appears to block division and giving rise to
elongated cells in Enterococcus hirae (Higgins et al., 1980),
indicating that membrane lipid composition may also be
important for cell division in this organism.
Proper localization of the FtsZ ring is followed by the
recruitment of other division proteins at mid cell, including
the division-specific PBPs (Errington et al., 2003; Goehring &
Beckwith, 2005). PBPs were initially thought to localize by
direct or indirect protein–protein interaction with the FtsZ ring.
However, substrate recognition may also operate in recruiting
PBPs to the septum. Two lines of evidence point towards the
existence of such a mechanism. The first is the role of the
DD-carboxypeptidase PBP3 in co-ordinating the division
process in Streptococcus pneumoniae. By trimming the pentapeptides, PBP3 degrades the substrate of the transpeptidase
activity of other PBPs (Severin et al., 1992; Morlot et al.,
2004a, b). Before division starts, PBP3 appears to be distributed over the whole cell surface, except at the equator
(Morlot et al., 2004a, b). This distribution of PBP3 would
permit the presence of pentapeptides only at the equator, to
where other PBPs could be recruited by affinity for their
substrate. Interestingly, a Streptococcus pneumoniae mutant
defective in PBP3 shows aberrant morphology and defects in
division, with multiple aborted septa (Schuster et al., 1990),
and in the colocalization of PBPs with the FtsZ ring (Morlot
et al., 2004a, b). The reason for the exclusion of PBP3 from
the equator is unknown, but the lipid composition at the
division site might conceivably influence the localization of
PBP3, as this protein is thought to be attached to the
membrane by a C-terminal amphipathic helix lying in the
outer-leaflet of the lipid bilayer (Morlot et al., 2004a, b).
The second line of evidence came from observations that in
Staphylococcus aureus, proper localization of the single HMW
class A PBP2 was lost after treatment with a b-lactam, which
inactivates the enzyme active site. The D-Ala-D-Ala dipeptide at
the free end of the stem peptides of the peptidoglycan or of its
precursor lipid II is the donor substrate of the transpeptidation
reaction catalyzed by PBPs. Proper localization of PBP2 was
also lost when the presence of D-Ala-D-Ala was abolished by
addition of D-cycloserine or when the access of PBPs to the DAla-D-Ala moieties was blocked by vancomycin (Pinho &
Errington, 2005). Substrate recognition may also contribute to
the localization of the PBPs in noncocci organisms, as in
Escherichia coli treatment with a b-lactam thought to be specific
for the septal class B PBP3 abrogates the septal localization of
this PBP (Wang et al., 1998).
Note that substrate-mediated localization may partly
solve the following problem of how the low-affinity PBPs
2008 Federation of European Microbiological Societies
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c
356
(PBP2a in methicillin-resistant Staphylococcus aureus or
PBP5 in enterococci) can replace other PBPs when these are
inhibited by b-lactams but are still present. Inhibited PBPs
may not find their proper localization, leaving their place
within machineries to the low-affinity PBPs. The low-affinity
PBPs, in turn, may recruit the required class A PBPs, such as
PBP2 in Staphylococcus aureus (Pinho & Errington, 2005).
An interesting finding from immunofluorescence studies
in Streptococcus pneumoniae was the existence of a delay
between constriction of the FtsZ ring and constriction of the
distribution of the cell wall synthesis machineries (Morlot
et al., 2003, 2004a, b). Similarly, FtsZ is relocated to the
future division site before PBPs and FtsW. In rods, the FtsZ
ring is also assembled at mid cell at the onset of the division
process, some time before the recruitment of other components, which can be enrolled as pre-exisiting subcomplexes
(e.g. FtsQ/DivB, FtsL, FtsB/DivIC, Den Blaauwen et al.,
2008, and references therein). The observation that the
septal peptidoglycan synthesis machinery and FtsZ are not
strictly colocalized throughout the cell cycle showed that coordination of the diverse aspects of division is not achieved
through the constitution of a super complex comprising all
the division proteins, which would remain together during
the whole septation process. Note that the delay between
constriction of the FtsZ ring and the septal cell wall assembly
machinery may differ in various species. Cryo-electron
microscopy in Enterococcus gallinarum has shown that the
leading edge of the growing septum is very close to a
structure identified as the FtsZ ring, separated only by the
plasma membrane. In contrast, in Streptococcus gordonii, the
invaginating membrane, presumably following the constriction of the FtsZ ring, is largely decoupled and in advance
of the synthesis of the septal wall (Zuber et al., 2006).
Besides FtsZ, FtsA is the only conserved essential division
protein to be cytoplasmic, although it is associated with the
membrane. The cellular localization of FtsA in rods parallels
that of FtsZ, it associates with FtsZ and a proper ratio of
FtsA to FtsZ molecules is required for efficient division
(Errington et al., 2003; Den Blaauwen et al., 2008). An
intriguing effect of an FtsA mutant in Escherichia coli on the
reactivity of the division specific PBP3 with penicillin has
suggested a role of FtsA in cell wall synthesis during division
(Tormo et al., 1986). In Streptococcus pneumoniae, FtsA was
shown by immunofluorescence and immuno-gold electron
microscopy to adopt a localization similar to that of FtsZ
(Lara et al., 2005). Most interestingly, recombinant pneumococcal FtsA was found to polymerize in vitro, unlike FtsA
from other microorganisms (Lara et al., 2005). The significance of this finding remains unknown, as is the precise
function of FtsA.
Finally, septum closure has to be tightly regulated with
chromosome segregation to avoid chromosome breakage. In
the absence of a nucleoid occlusion effect in some cocci, it is
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A. Zapun et al.
possible that an orthologue of the division protein FtsK/
SpoIIIE has a particularly important role in co-ordinating
septum closure and chromosome segregation. This protein
has a cytoplasmic domain with DNA translocase activity and
is of particular importance during sporulation of B. subtilis, a
process that requires packaging of the DNA in a much smaller
compartment than during vegetative growth. Some cocci cells
are not much larger than a Bacillus spore. Therefore, a DNA
pump may be essential in some cocci for successful chromosome segregation before septum completion.
Open questions
The models presented above for peptidoglycan metabolism
and its role in cell morphogenesis do not address some
important aspects of this process. The first aspect is cell wall
thickening of ovococci. The careful measurements of dividing Enterococcus hirae by Higgins & Shockman (1976)
showed that both septal and peripheral walls, in addition
to increasing their surfaces, are also thickened during the cell
cycle. Strictly localized machineries cannot account for such
thickening. One proposal is that enzymatic complexes
involved in division are leaving the leading edge of the
septum progressively as it closes. These machineries could
add new layers of material to the inner face of the cell wall
while drifting from the septum edge to be relocated at the
equator. The drift could be a random diffusion, with
the machineries reaching the equator being trapped by the
presence of FtsZ. The possibility of additional layering
synthesis is attractive, as it would also explain the fact that
the cell shape, cell volume and cell surface are constant at
various growth rates, but that the amount of synthesized
peptidoglycan is not (Edelstein et al., 1980). The thicker cell
wall of slow-growing cells would result from the longer time
available for the PBPs to relocate at the equator of ovococci.
The second aspect is related to peptidoglycan hydrolases.
Although these proteins are essential for proper peptidoglycan metabolism, the precise function of each of the many
hydrolases present in different bacteria is still unknown,
including the identity of the enzyme(s) responsible for
splitting the septum, neither is it known in cocci as to which
(if any) hydrolases are part of the multienzymatic complexes
responsible for cell wall synthesis. In Staphylococcus aureus,
the major autolysin is encoded by the atl gene, which
encodes a precursor protein that undergoes cleavage to
generate a mature amidase and glucosaminidase (Oshida
et al., 1995). These proteins localize at the septal region,
which is in agreement with their proposed function in the
hydrolysis of peptidoglycan for separation of daughter cells
after division (Yamada et al., 1996). However, Staphylococcus
aureus genome encodes for 16 other peptidoglycan hydrolases (S. Foster, pers. commun.) and the function of the
majority of them remains unclear.
FEMS Microbiol Rev 32 (2008) 345–360
357
Coccal morphogenesis
The enzyme that splits the septal cell wall of streptococci is
possibly PcsB. Inactivation of the gene encoding PcsB in
Streptococcus agalactiae and Streptococcus mutans produces cells
that grow in clumps, with unsplit cross-wall joining adjacent
cells and other size and shape aberrations (Chia et al., 2001;
Reinscheid et al., 2001). In Streptococcus pneumoniae, the pcsB
gene is essential, but reduced expression also generates joined
cells with abnormal morphologies (Ng et al., 2004). PcsB
contains a CHAP domain (for cysteine, histidine-dependent
amidohydrolase/peptidase). However, a degradative biochemical
activity of PcsB on the peptidoglycan has not been demonstrated to date. Although PcsB orthologues are found in
streptococci and Lactococcus lactis, a true homologue of PcsB is
absent from enterococci. Instead, enterococci have a protein
named SagA [Enterococcus faecium; (Teng et al., 2003)], SagBb
(Enterococcus hirae) or SalA (Enterococcus faecalis). The Nterminal parts of these proteins and PcsB are similar. In contrast,
the C-terminal parts of SagA/Bb and SalA are not CHAP
domains but belong to other distantly related groups of the
NlpC/P60 superfamily of enzymes (Anantharaman & Aravind,
2003). The similar chromosomal environment of the genes encoding PcsB, SagA/Bb and SalA suggests that they perform the
same function, although some domain shuffling has occurred,
possibly in relation to diverse peptidoglycan composition.
Note that in Streptococcus pneumoniae, once the septum is
split, cells remain attached by the tip of their poles and form
long chains without the action of a peptidoglycan hydrolase
termed LytB, which is required for cell separation (De Las
Rivas et al., 2002). Another hydrolase termed LytA, which is
responsible for the spontaneous lysis of Streptococcus pneumoniae, may play a minor role in cell separation as the
deletion of lytA causes the formation of short chains of six to
eight cells (Sanchez-Puelles et al., 1986).
Lastly, much has been speculated about the existence of
multienzymatic complexes responsible for cell wall synthesis
in bacteria. However it is still not known as to which enzymes
are part of such complexes, or how they are regulated in time
and space. Most studies regarding the composition of such
complexes have been performed in either Escherichia coli or B.
subtilis, both of which have a large number of PBPs. Owing to
the lower number of PBPs, cocci are probably better model
organisms for these studies, which are essential for understanding the process of cell wall synthesis. This knowledge is
important not only in the context of cell morphogenesis and
division but also in the context of antibiotic resistance, as cell
wall synthesis is the target of a large number of very effective
antimicrobial agents.
The valuable insights into bacterial morphogenesis that
have been gained by a limited number of investigations in
cocci indicate that pursuing comparative studies of morphologically distinct organisms, including clinically relevant
pathogens, is the way ahead to gain a better understanding
of how bacteria grow and divide.
FEMS Microbiol Rev 32 (2008) 345–360
Acknowledgements
Work in the Vernet’s laboratory is supported by grants from
the 6th European Framework Program (COBRA LSHM-CT2003-503335 and EUR-INTAFAR LSHM-CT-2004-512138).
Work in M. Pinho’s laboratory is supported by grant POCI/
BIA-BCM/56493/2004 from Fundação para a Ciência e
Tecnologia. The authors thank Anne Marie Di Guilmi for
the thin section of pneumococcus. The authors are grateful
to Orietta Massidda for pointing out the difficulties with the
immunofluorescence data and to Sergio Filipe and Dirk-Jan
Scheffers for critically reading the manuscript.
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